Persistent and chronic infections are often refractory to antibiotics due to antibiotic tolerance of a subpopulation of cells that are not antibiotic resistant mutants, but rather are dormant cells that survive antibiotic killing. Our findings show that Pseudomonas aeruginosa and Burkholderia species excrete a small molecule that serves as a persistence infochemical that signals the accumulation of these antibiotic tolerant persister (AT/P) cells and changes that are critical for pathogen adaptation and important for chronic infection. Our aim is to achieve a paradigm shift in persistent infection interventions by introducing a treatment that disrupts the bacterial signaling that induces AT/P cell formation using compounds we have identified; to achieve this aim we will refine and validate lead compounds in vivo, using adapted mouse models of infection. Our approach is fundamentally different from traditional antimicrobial therapies as it specifically targets the AT/P subpopulation of cells that survive antibiotic treatment (and host defense killing mechanisms), and that are ultimately responsible for persistent and relapsing infections. We propose to develop this approach through experiments employing P. aeruginosa, a recalcitrant Gram-negative bacterium that defies eradication by antibiotics, forms biofilms, and exemplifies current clinical problematic pathogens. In the R21 phase Aim 1 studies, we will use structure-activity relationship (SAR) data to refine the chemical compositions of the particularly promising 1st generation compounds we have identified. The feasibility of this approach has been established by our prior generation of a series of structurally related agents that block the synthesis of a pr-AT/P signaling molecule and reduce virulence in vivo. In Aim 2, we will perform a series of microbiological, cellular, and biochemical evaluations of the 2nd generation compounds to assess their relative IC50 values and their efficacy against several clinical isolates (including pan-resistant and multi-drug resistant isolates) when used in combination with different classes of antibiotics or alone, as well as their ability to disrupt the synthesis of the signaling molecule and prevent the resultant imbalance in DNA topology and translational effects that we have demonstrated to occur in cells that have transitioned to the AT/P state. The R33 phase (Aims 3 and 4) will be undertaken if our well-defined milestones are achieved. In Aim 3, we will assess the pharmacological efficiency properties of the most promising 2nd generation compounds identified in the R21 phase. In Aim 4, the compounds' efficacies against drug resistant, tolerant pathogens that co-exist with P. aeruginosa in human infections and similarly form AT/P cells (i.e. Acinetobacter baumannii, Klebsiella pneumoniae, and Burkholderia species) will be tested in mono- and polymicrobial planktonic and biofilm settings. Combination drug assays will be performed to determine whether our lead molecules improve antibiotic clearance of biofilms. Highly prioritized advanced leads will then be validated in established mouse models that we have developed. The overall goal of these studies is to carefully assess the potential utility of lead small molecules that target AT/P cells as a new way to intervene against chronic and persistent infections that have thus far been untreatable. These anti-AT/P molecules may be combined with traditional antibiotic therapies for optimal effectiveness.